Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: ( a ) Representative Western blot labelled with anti-NICD and anti-GAPDH (loading control) antibodies for HMVEC-Cs exposed to 10 dyn.cm −2 laminar shear stress (SS) for 1 hr. Static was without SS. Cells were transfected with control siRNA (siCtrl) or Piezo1 siRNA (siPiezo1). The expected mass of NICD is 110 kDa. Lower molecular bands were also apparent in some experiments and may have been degraded NICD. ( b ) Quantification of data of the type exemplified in ( a ), showing mean ± SD data for abundance of NICD normalized to siCtrl Static (n = 4). ( c ) Representative Western blot labelled with anti-NICD and anti-GAPDH antibodies for HMVEC-Cs treated for 30 min with 0.2 µM Yoda1 or vehicle (DMSO) after transfection with control siRNA (siCtrl) or Piezo1 siRNA (siPiezo1). ( d ) Quantification of data of the type exemplified in ( c ), showing mean ± SD for abundance of NICD normalized to siCtrl DMSO (n = 3). Statistical analysis: Two-way ANOVA test was used, indicating *p<0.05, **p<0.01 or not significantly different (NS). Figure 1—source data 1. Source data for .

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Western Blot, Control, Shear, Transfection

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: ( a ) Summarized mean ± SD (n = 3) quantitative PCR data for fold-change in Piezo1 mRNA in HMVEC-Cs treated for 2 hr min with 0.2 mM Yoda1 or vehicle (DMSO) after transfection with control siRNA (siCtrl) or Piezo1 siRNA (siPiezo1). ( b ) Representative Western blot labelled with anti-VEGRF2, anti-PECAM-1 (anti-CD31) and anti-GAPDH antibodies for HMVEC-Cs after transfection with control siRNA (siCtrl) or Piezo1 siRNA (siPiezo1). ( c, d ) Quantification of data of the type exemplified in ( b ), showing mean ± SD data for abundance of VEGFR2 ( c ) and PECAM-1 ( d ) normalized to siCtrl (n = 3). ( e–f ) Representative intracellular Ca 2+ measurement traces ( e ) in HMVEC-Cs during application of 2 μM Yoda1 or its control DMSO, 48 hr after transfection with control siRNA (siCtrl) or Piezo1 siRNA (siPiezo1), shown as mean ± SD of the amplitude for n = 3 ( f ). Two-way ANOVA test was used for ( a ) and t-test for ( c, d, f ), indicating **p<0.01, ***p<0.001 or not significantly different (NS). Figure 1—figure supplement 2—source data 1. Source data for .

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Real-time Polymerase Chain Reaction, Transfection, Control, Western Blot

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: ( a ) Representative Western blot labelled with anti-NICD and anti-GAPDH antibodies for HMVEC-Cs treated for 30 min with 0.2 µM Yoda1 or vehicle (DMSO) in the absence or presence of 10 µM DAPT. ( b ) Quantification of data of the type exemplified in ( a ), showing mean ± SD data for abundance of NICD normalized to vehicle (DMSO) control (n = 3). ( c–d ) Representative intracellular Ca 2+ measurement traces ( c ) in HMVEC-Cs during prolonged application of 0.5 μM Yoda1 or its vehicle control (DMSO) simultaneously with 10 mM DAPT or its vehicle control (DMSO), shown as mean ± SD of the area under the curve for n = 3 ( d ). Two-way ANOVA test was used for ( b ) and t-test for ( d ), indicating *p<0.05, **p<0.01, ***p<0.001 or not significantly different (NS). Figure 1—figure supplement 4—source data 1. Source data for .

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Western Blot, Control

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: ( a ) ADAM10 enzyme activity assessed by specific peptide degradation and subsequent fluorescence emission after 30 min exposure of HMVEC-Cs to 10 dyn.cm −2 laminar shear stress (SS). Static was without SS. Cells were transfected with control siRNA (siCtrl) or Piezo1 siRNA (siPiezo1). Data are shown as mean ± SD data (n = 3) relative to static condition. ( b ) ADAM10 enzyme activity assessed after 30 min treatment of HMVEC-Cs with 0.2 µM Yoda1 in the absence or presence of 5 µM GI254023X (GI). Data are shown as mean ± SD data (n = 4) relative to DMSO condition. ( c, d ) Quantification of uncleaved (95 kDa) and cleaved (62–64 kDa) ADAM10 in HMVEC-Cs after treatment for 30 min with Yoda1 (0.2 µM). The 85 kDa band between the uncleaved and cleaved ADAM10 was non-specific labelling not related to ADAM10 . Data represent mean ± SD (n = 3) and normalization was to the reference protein, GAPDH. ( e ) Example Western blot labelled with anti-NICD and anti-GAPDH antibodies for HMVEC-Cs treated for 30 min with 0.2 µM Yoda1 or vehicle (DMSO) in the absence or presence of 5 µM GI254023X (GI). ( f ) Quantification of data of the type exemplified in ( e ), showing mean ± SD data for abundance of NICD normalized to DMSO (n = 3). ( g ) Representative Western blot labelled with anti-NICD and anti-GAPDH antibodies for HMVEC-Cs treated for 30 min with 0.2 µM Yoda1 or vehicle (DMSO) after transfection with control siRNA (siCtrl) or ADAM10 siRNA (siADAM10). ( h ) Quantification of data of the type exemplified in ( g ), showing mean ± SD data for abundance of NICD normalized to siCtrl DMSO (n = 3). Statistical analysis: Two-way ANOVA test was used for ( a, b, f, h ), indicating *p<0.05, **p<0.01, ***p<0.001; t-Test was used for ( d ), indicating **p<0.01; NS, not significantly different. Figure 2—source data 1. Source data for .

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Activity Assay, Fluorescence, Shear, Transfection, Control, Western Blot

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: ( a ) Summarized mean ± SD (n = 3) quantitative PCR data for fold-change in ADAM10 mRNA in HMVEC-Cs treated for 2 hr with 0.2 µM Yoda1 or vehicle (DMSO) after transfection with control siRNA (siCtrl) or ADAM10 siRNA (siADAM10). ( b ) Representative Western blot labelled with anti-ADAM10 antibody for HMVEC-Cs treated for 30 min with 0.2 µM Yoda1 after transfection with control siRNA (siCtrl) or ADAM10 siRNA (siADAM10). ( c, d ) Quantification of data of the type exemplified in ( b ), showing mean ± SD data for abundance of uncleaved ADAM10 (95 kDa) ( c ) and non-specific labelling of an unknown protein (85 kDa) ( d ) normalized to siCtrl (n = 3). ( e–f ) Representative intracellular Ca 2+ measurement traces ( e ) in HMVEC-Cs during application of 2 μM Yoda1 or its vehicle control (DMSO), 48 hr after transfection with control siRNA (siCtrl) or ADAM10 siRNA (siADAM10), shown as mean ± SD of the amplitude for n = 3 ( f ). ( g–h ) Representative intracellular Ca 2+ measurement traces ( g ) in HMVEC-Cs during prolonged application of 0.5 μM Yoda1 or its vehicle control (DMSO) simultaneously with 5 µM GI254023X (GI) or its vehicle control (DMSO), shown as mean ± SD of the area under the curve for n = 3 ( h ). ( i ) Representative Western blot labelled with anti-NICD and anti-GAPDH antibodies for HMVEC-Cs treated for 30 min with 0.2 µM Yoda1 or vehicle (DMSO) in the absence or presence of 0.5 µM GI254023X (GI) ( j ) Quantification of data of the type exemplified in ( i ), showing mean ± SD data for abundance of NICD normalized to siCtrl DMSO (n = 3). ( k ) ADAM10 enzyme activity assessed by specific peptide degradation and subsequent fluorescence emission after 30 min treatment of HMVEC-Cs with 0.2 µM Yoda1 in the absence or presence of 30 µM Gd 3+ . Data are shown as mean ± SD data (n = 3) relative to vehicle condition. Statistical analysis: Two-way ANOVA test was used for ( a, c, d, j, k ), indicating *p<0.05, **p<0.01 and ***p<0.001. T-test was used for ( f, h ), indicating ***p<0.001. NS indicates no significant difference. Figure 2—figure supplement 2—source data 1. Source data for .

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Real-time Polymerase Chain Reaction, Transfection, Control, Western Blot, Activity Assay, Fluorescence

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: ( a, b, c ) Summarized mean ± SD (n = 3) quantitative PCR data for fold-change in HES1 ( a ), DLL4 ( b ) and HEY1 ( c ) mRNA in HMVEC-Cs treated for 2 hr with 0.2 µM Yoda1 or vehicle (DMSO) after transfection with control siRNA (siCtrl) or Piezo1 siRNA (siPiezo1). ( d, e, f ) Summarized mean ± SD (n = 3) quantitative PCR data for fold-change in HES1 ( d ), DLL4 ( e ) and HEY1 ( f ) mRNA in HMVEC-Cs treated for 2 hr with 0.2 µM Yoda1 in the absence or presence of 10 µM DAPT. ( g, h, i ) Summarized mean ± SD (n = 3) quantitative PCR data for fold-change in HES1 ( g ), DLL4 ( h ) and HEY1 ( i ) mRNA in HMVEC-Cs treated for 2 hr with 0.2 µM Yoda1 or vehicle (DMSO) after transfection with control siRNA (siCtrl) or ADAM10 siRNA (siADAM10). ( j, k, l ) Summarized mean ± SD (n = 3) quantitative PCR data for fold-change in HES1 ( j ), DLL4 ( k ) and HEY1 ( l ) mRNA in HMVEC-Cs treated for 2 hr with 0.2 µM Yoda1 in the absence or presence of 5 µM GI254023X (GI). Normalization and statistical analysis: mRNA expression was normalized to GAPDH mRNA abundance. Two-way ANOVA test was used, indicating *p<0.05, **p<0.01, ***p<0.001 or not significantly different (NS). Figure 3—source data 1. Source data for .

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Real-time Polymerase Chain Reaction, Transfection, Control, Expressing

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: Additional gene quantification for HMVEC-Cs treated for 2 hr with 0.2 μM Yoda1 or vehicle (DMSO) ( a ) HEY2. ( b ) HES2. ( c ) JAG1 . Statistical analysis: t-test was used. Figure 3—figure supplement 1—source data 1. Source data for Figure 3—figure supplement 1 .

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques:

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet: Activation of Piezo1 channel by mechanical force (e.g. fluid flow) or chemical agonist (Yoda1) causes elevation of the intracellular Ca 2+ concentration which stimulates enzymatic activity of ADAM10 to cause S2 and S3 Notch1 cleavage and release of NICD to drive target gene expression that includes increased expression of Hes1, Dll4, Hey1, HeyL, Jag1, Hes2, Hes3 and Efnb2 in hepatic vasculature of the mouse. In the schematic we also include the suggested contributions from ADAM10/Notch1/NICD independent signalling from Piezo1 and non-canonical Notch1 signalling via the Notch1 transmembrane domain (TMD), as described and referenced in the text.

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Activation Assay, Concentration Assay, Activity Assay, Targeted Gene Expression, Expressing

Journal: eLife

Article Title: Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells

doi: 10.7554/eLife.50684

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Yoda1 , Tocris Bioscience , Cat# 5586–10 , .

Techniques: Cell Culture, Sequencing, Control, Activity Assay

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Piezo1 mediates the suppression of osteogenic gene expression under low‐strain CTS. The knockdown efficiency was confirmed to be approximately 80% for each gene. (A) RT‐qPCR showed that si Piezo1 reversed the low‐strain CTS‐induced downregulation of Runx2 and Osx . (B, C) si Piezo2 or si Trpv4 did not reverse osteogenic gene suppression by low‐strain CTS. An ordinary one‐way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test was used. Each group was compared with the siN/ C without the CTS group. Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Gene Expression, Knockdown, Quantitative RT-PCR

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Activation of Piezo1 by Yoda1 suppresses osteogenic markers in rat AF cells. (A) RT‐qPCR showed downregulation of Runx2 , Osx , and Alp after treatment with 10 μM Yoda1 for 12 h. (B) Western blotting analysis revealed that RUNX2 protein levels decreased in rat AF cells after 24‐h Yoda1 treatment. Figure shows the full‐length blot images. Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Activation of Piezo1 by Yoda1 suppresses osteogenic markers in human AF cells. Human AF cells were isolated from nondegenerated (Pfirrmann grade 1) and severely degenerated (Pfirrmann grade 5) intervertebral discs. (A) PIEZO1 mRNA expression showed no significant difference between grade 1 and grade 5 cells. (B–E) Consistent with observations in rat AF cells, treatment with Yoda1 (10 μM, 12 h) significantly downregulated osteogenesis‐related genes ( RUNX2 , OSX ) at the mRNA level and reduced RUNX2 protein expression. Full‐length Western blot images are shown in Figure . Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Isolation, Expressing, Western Blot

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Transcriptomic analysis of AF cells following Piezo1 activation by Yoda1. (A) PCA showed a clear separation between the control (DMSO) and Yoda1‐treated groups. (B) Volcano plot of DEGs shows 3281 upregulated and 3362 downregulated genes. (C) GSEA reveals enrichment of ossification‐ and calcium signaling‐related pathways, including regulation of ossification, regulation of bone mineralization, and calcineurin‐mediated signaling, in Yoda1‐treated AF cells.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Control

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Piezo1 activation inhibits BMP2‐induced osteogenesis through calcineurin signaling. (A) Schematic of the experimental schedule for the BMP2 and Yoda1 treatment. (B) RT‐qPCR showed that Yoda1 cotreatment suppressed the BMP2‐induced upregulation of Osx and Alp in AF cells. (C, D) Alizarin Red staining revealed that BMP2 alone enhanced calcium deposition, whereas cotreatment with Yoda1 reduced this staining intensity. (E) Calcineurin phosphatase assay showing that Yoda1 significantly increased calcineurin enzymatic activity compared with DMSO control; this effect was abolished by co‐treatment with cyclosporin A (CsA). (F, G) Immunocytochemistry for p‐Smad1/5/9 showed increased nuclear translocation after BMP2 treatment but significantly decreased BMP2 + Yoda1 cotreated group. Furthermore, cotreatment with the calcineurin inhibitor CsA rescues the nuclear translocation of p‐Smad1/5/9. Statistical significance was set at P < 0.05, with significance levels denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Activation Assay, Quantitative RT-PCR, Staining, Phosphatase Assay, Activity Assay, Control, Immunocytochemistry, Translocation Assay

Journal: JOR Spine

Article Title: Mechanosensitive Ion Channel PIEZO1 Suppresses BMP2 ‐Induced Ossification of the Annulus Fibrosus Cells

doi: 10.1002/jsp2.70168

Figure Lengend Snippet: Proposed mechanism schema. Piezo1‐mediated calcium influx activates calcineurin, which dephosphorylates p‐Smad1/5/9, inhibiting its nuclear translocation and BMP2 signaling.

Article Snippet: We measured intracellular calcium responses using the calcium indicator dye Fluo‐8 NW (AAT Bioquest, Pleasanton, CA, Cat. #36315) and PIEZO1 agonist Yoda1 (Selleck, Houston, TX, Cat. #S6678).

Techniques: Translocation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Piezo type mechanosensitive ion channel component 1 facilitates gastric cancer omentum metastasis

doi: 10.1111/jcmm.16217

Figure Lengend Snippet: Piezo1 induces cell proliferation and inhibits cell apoptosis in GC cells. A‐C, qRT‐PCR and Western blot assays were used to detect the expression of Piezo1 in GC cells transfected with two siRNAs. D, E, CCK‐8 assay results showed the cell viability of two GC cells treated with different concentrations of Yoda1. F, G, The effects of Piezo1 on colony formation ability were observed in two GC cells transfected with si‐Piezo1 or Yoda1. H, I, Flow cytometry assay was used to detect the cell apoptosis in two GC cells treated with different concentrations of Yoda1. J‐L, Western blot was performed to detect the expression of P53, P21, CDK4, CDK6 and CyclinD1 proteins in two GC cells transfected with si‐Piezo1. M, N, Immunohistochemistry results showed the decreased Ki‐67‐positive cells following transfection with si‐Piezo1. * P < .05; ** P < .01; *** P < .001; **** P < .0001

Article Snippet: Cells were treated with different concentrations of Yoda1 (12.5, 50, 100, 200 and 400 μmol/L; Cayman Chemical, #448947‐81‐7, USA) for 48 hours.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, CCK-8 Assay, Flow Cytometry, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Piezo type mechanosensitive ion channel component 1 facilitates gastric cancer omentum metastasis

doi: 10.1111/jcmm.16217

Figure Lengend Snippet: Piezo1 promotes Ca 2+ level and mitochondrial membrane potential in GC cells. A‐C, Fluo4 assay results showed the Ca 2+ level in SNU‐1 and HGC‐27 cells transfected with Yoda1 or si‐Piezo1. D‐F, JC‐1 assay results suggested the mitochondrial membrane potential in two GC cells after treatment with Yoda1 or si‐Piezo1. * P < .05; ** P < .01; **** P < .0001

Article Snippet: Cells were treated with different concentrations of Yoda1 (12.5, 50, 100, 200 and 400 μmol/L; Cayman Chemical, #448947‐81‐7, USA) for 48 hours.

Techniques: Membrane, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Piezo type mechanosensitive ion channel component 1 facilitates gastric cancer omentum metastasis

doi: 10.1111/jcmm.16217

Figure Lengend Snippet: Piezo1 promotes the expression of HIF‐1α and VEGF proteins in GC cells. A, B, Representative images of immunohistochemistry for SNU‐1 and HGC‐27 two GC cells treated with Yoda1 and si‐Piezo1. C, D, The expression of Piezo1, HIF‐1α and VEGF was quantified in GC cells according to immunohistochemistry. E‐H, Immunofluorescence assay results showed the expression of Piezo1, HIF‐1α and VEGF in GC transfected with Yoda1 and si‐Piezo1. * P < .05; ** P < .01; *** P < .001; **** P < .0001

Article Snippet: Cells were treated with different concentrations of Yoda1 (12.5, 50, 100, 200 and 400 μmol/L; Cayman Chemical, #448947‐81‐7, USA) for 48 hours.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Piezo type mechanosensitive ion channel component 1 facilitates gastric cancer omentum metastasis

doi: 10.1111/jcmm.16217

Figure Lengend Snippet: Piezo1 could promote cell migration via up‐regulation of HIF‐1α in GC cell. A, B, Representative images of SNU‐1 and HGC‐27 two GC cells stably transfected with HIF‐1α and Piezo1 knockdown. C, D, Western blot was used to assess the transfection efficiency of HIF‐1α knockdown in GC cells. E‐H, Transwell assay was performed to detect the migrant ability of GC cells after treatment with si‐Piezo1, Yoda1 or si‐Piezo1 + Yoda1. I‐L, Wound healing assay was used to measure the wound distance for GC cells transfected with si‐Piezo1, Yoda1 or si‐Piezo1 + Yoda1. * P < .05; ** P < .01; **** P < .0001

Article Snippet: Cells were treated with different concentrations of Yoda1 (12.5, 50, 100, 200 and 400 μmol/L; Cayman Chemical, #448947‐81‐7, USA) for 48 hours.

Techniques: Migration, Stable Transfection, Transfection, Knockdown, Western Blot, Transwell Assay, Wound Healing Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the proposed mechanism of action of Yoda1 on PIEZO1 (left) and close-up view of the THU8–9 interface with previously proposed residues lining putative Yoda1 binding sites highlighted in sphere representation. (B) , time course of Ca 2+ -influx (F/F0) evoked by 10 and 100 µM Yoda1 in cells expressing PIEZO1 (left) and P1-A2094W (center) assessed by GCamp8 imaging together with comparison of maximum responses (right). (C) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) stretch-evoked currents by 30µM Yoda1. Example traces evoked by incrementing pressure stimuli (left), comparison of peak/sustained ratio (middle) using Mann-Whitney test (PIEZO1: CTL = 4.02, N=14 vs Yoda1 = 1.38, N=15, P=0.0000131) and Student’s t-test(P1.A2094W: CTL = 8.35, N=10 vs Yoda1 = 1.19, N=10, P=0.0033), pressure-response curves (i.e. peak current amplitude at indicated pressure normalized to maximal response, bottom right) and comparison of P 50 values in the absence and presence of Yoda1 using Student’s t-test (PIEZO1: CTL = 26.1 ± 8.9 mmHg, N=14 vs Yoda1 = -16.9 ± 6.65 mmHg, N=13, P=0.006) and Mann-Whitney test (P1.A2094W: CTL = -43.7 ± 10.04 mmHg, N=10 vs Yoda1 = -27.4 ± 6.7 mmHg, N=10, P=0.0005). (D) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) poking-evoked currents in whole-cell recordings by 30µM Yoda1. Example traces evoked by incrementing (Δ 800nm, left), comparison of inactivation time constants obtained with exponential decay fit (middle) using Mann-Whitney test (PIEZO1: CTL = 15.9 ± 4.3 ms, N=14 vs Yoda1 = 41.1 ± 21.6, N=12, P=0.000258) and Student’s t-test (P1.A2094W: CTL = 6.39 ± 2.8 ms, N=18 vs Yoda1 = 8.3 ± 2.7, N=20, P=0.0405), displacement-response curves (i.e. peak current amplitude vs. indicated stimulus magnitude; bottom, right) and comparison of mechanical activation thresholds using Mann-Whitney test (PIEZO1: CTL = 4.1 ± 1.2 µm, N=14 vs Yoda1 = 2.8 ± 1.4 µm, N=12, P=0.029; P1.A2094W: CTL = 4.8 ± 1.04 µm, N=18 vs Yoda1 = 3.8 ± 1.1 µm, N=20, P=0.0069).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Binding Assay, Expressing, Imaging, Comparison, MANN-WHITNEY, Activation Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Side view of curved (PDB:7WTL, top left) and flattened (PDB:7WTU, bottom left) PIEZO1 cryo-EM structure with close-up side (middle) and top (right) view of THU8–9 interfaces with the putative binding pockets detected by DoGSite3 algorithm shown in isomesh representation. Amino acid side chains that line the pockets are shown in stick representation. (B) , Close-up views of Yoda1 binding poses detected by AutoDock Vina in the curved (top) and flattened (bottom) PIEZO1 conformation. Note, only the binding poses with the highest scores that align with the binding pockets detected in (A) are shown. Residues that are within a distance of 3.5 Å of Yoda1 are shown in stick representation. Note, F1715 appears to be involved in Yoda1 binding in pocket-2 and pocket-3 in the flat conformation. (C) , AutoDock Vina detects multiple possible docking poses for Yoda1. The bar graph shows the percentage of docking poses in which the indicated amino acids are located within 3.5 Å of Yoda1. Note, F1715 is in close proximity of Yoda1 in 78% of all possible binding poses indicating an important role of F1715 in Yoda1 binding in the flat state.

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the overall strategy to resolve Yoda1 induced conformational changes (flattening) measured through changes in interblade distance. Insets illustrate the labelling of PIEZO1 with ALFA tag inserted after H86, and the DNA-PAINT method (left). Individual bound fluorophore is located with high precision in 3 dimensions via 3D-MINFLUX and its iterative process (middle), leading to multiple localisations of the same molecule. (B) Confocal image of a PIEZO1-ALFA-mGL expressing N2a-P1KO cell (left) and corresponding 3D-MINFLUX localizations (right), colored by Z range. Inset shows a triple-labelled PIEZO1, with the 3D scatter plots of the raw localizations and a superimposed cryo-EM structure. The 2D in-plane projections of the 3D data were fitted with a bivariate Gaussian distribution, with their probability densities, enabling determination of the average interblade distance (right). (C) Time-course of the average ± s.e.m. (from N=3-4 independent experiments) of the maximal Yoda1 (50µM) effect on the normalized fluorescence (F/F0) for PIEZO1 (top), A2094W (middle) and V1714A_F1715A (bottom). (D-F) In-plane projections of representative trimers examples of PIEZO1 ( D ), A2094W ( E ) and V1714A_1715A ( F ) from cells treated with cytochalasin-D (CTL) or with cytochalasin-D and Yoda1 (50µM) (left). Comparison of the mean ± s.e.m. interblade distance of the identified trimers after addition of Yoda1 for PIEZO1 ( D , N=93 and 110), A2094W ( E , N=61 and 64) and V1714A_1715A ( F , N=87 and 59), with unpaired t-test (right).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Expressing, Cryo-EM Sample Prep, Fluorescence, Comparison

Journal: Scientific Reports

Article Title: Enhanced Ca 2+ influx in mechanically distorted erythrocytes measured with 19 F nuclear magnetic resonance spectroscopy

doi: 10.1038/s41598-021-83044-z

Figure Lengend Snippet: 19 F NMR spectra of RBCs loaded with 5FBAPTA and treated with Yoda1 in the presence of Ca 2+ , at 37 °C. The peak from free 5FBAPTA inside the RBCs (initially 4 mmol [L RBC] −1 ) is highlighted in pink; yellow highlights the peak from the extracellular 5FBAPTA-calcium complex; green, the peak from the intracellular 5FBAPTA-calcium complex; and blue, the peak from the intracellular protein-5FBAPTA-calcium complex. The sample was 0.5 mL RBCs ( Ht = 0.73) in 154 mM NaCl and 10 mM d -glucose. The spectra were recorded every 10 min with NMR settings as for Fig. . The superimposed red spectrum at 7 min is from the 6th spectrum of a 1 h time course recorded with the RBCs in the presence of 2.0 µL 1 M CaCl 2 (corresponding to 4.0 mM Ca 2+ concentration averaged over the sample). Then, Yoda1 was added as 1.0 µL of 14 mM in DMSO; this value combined with the knowledge of the Ht value gave a concentration of 38 µmol [L RBC] −1 . Left inset: combined integral of the peak from 5FBAPTA-Ca (green plus yellow). Right inset: integral of intracellular free 5FBAPTA (pink). The fitted equations and their parameter values were (left and right, respectively): 1.06 ± 0.07 + (0.011 ± 0.003) t − (0.00005 ± 0.0003) t 2 , and 2.81 ± 0.09 − (0.010 ± 0.004) t − (0.00005 ± 0.0004) t 2 .

Article Snippet: Key reagents and their sources were: 5FBAPTA, Biotium (Landing Parkway, CA, USA); Yoda1, Glixx Laboratories (Hopkinton, MA, USA); and A23187, Sigma (Merck, Darmstadt, Germany).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Enhanced Ca 2+ influx in mechanically distorted erythrocytes measured with 19 F nuclear magnetic resonance spectroscopy

doi: 10.1038/s41598-021-83044-z

Figure Lengend Snippet: 19 F NMR spectra showing A23187 (and Yoda1 in comparison) stimulated uptake of Ca 2+ in RBCs loaded with 5FBAPTA (4 mmol [L RBC] −1 ), at 37 °C. Pink highlights the resonance corresponding to intracellular free 5FBAPTA that was assigned the chemical shift δ = 0.0 ppm; and the green highlights the peak from the calcium complex that was centred at ~ 5.8 ppm. The sample of 0.5 mL RBCs ( Ht = 0.62) was constituted in 154 mM NaCl, 10 mM glucose. ( a ) The RBCs had been loaded with 4 mM 5FBAPTA as described in Methods. Then, for ( b , c ), added with brisk mixing (by five-fold rapid inversion and re-inversion of the NMR tube) were: 5 µL 1 M CaCl 2 making the concentration 10 mM averaged over the volume of the sample; and 0.5 µL 20 mM A23187 in DMSO giving a concentration of 32 µmol (L RBC) −1 . For ( d ) 5 µL 1 M CaCl 2 and 1.0 µL 14 mM Yoda1 in DMSO giving a concentration of 45 µmol (L RBC) −1 were mixed into the 0.5 mL susupension. The time indicated on the right of each spectrum was the mid-point of spectral accumulation after a 2 min lag between mixing the sample and starting FID accumulation. NMR settings were as for Fig. except the time per spectrum was 10 min 47 s.

Article Snippet: Key reagents and their sources were: 5FBAPTA, Biotium (Landing Parkway, CA, USA); Yoda1, Glixx Laboratories (Hopkinton, MA, USA); and A23187, Sigma (Merck, Darmstadt, Germany).

Techniques: Comparison, Concentration Assay

Journal: Scientific Reports

Article Title: Enhanced Ca 2+ influx in mechanically distorted erythrocytes measured with 19 F nuclear magnetic resonance spectroscopy

doi: 10.1038/s41598-021-83044-z

Figure Lengend Snippet: Graphical representation of an RBC under strain in a stretched gel (see below); it is loaded with the Ca 2+- sensing chelator 5FBAPTA that yields separate 19 F NMR signals from the free and Ca 2+ -complexed forms. Ca 2+ enters via the mechanosensitive cation channel Piezo1 that can be activated (+ symbol) by the small-molecule compound Yoda1. Ca 2+ entry into the RBC can also be mediated by the Ca 2+ -selective ionophore A23187. [1,6- 13 C] d -glucose enters the cell via the glucose transporter GLUT1; it was used in conjunction with 13 C NMR spectroscopy to measure glycolytic flux under various experimental conditions. The model of the distorted RBC is based on Cartesian translation in Mathematica with the shape defined by the parametric equations given in .

Article Snippet: Key reagents and their sources were: 5FBAPTA, Biotium (Landing Parkway, CA, USA); Yoda1, Glixx Laboratories (Hopkinton, MA, USA); and A23187, Sigma (Merck, Darmstadt, Germany).

Techniques: Structural Proteomics